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library("devtools")
install_github("jw156605/SLICER")

Skipping install of 'SLICER' from a github remote, the SHA1 (cb1be8ac) has not changed since last install.
  Use `force = TRUE` to force installation

library(Seurat)
library(readr)
library(tidyverse)
library(HGC) 
library(latticeExtra)
library(lattice) 
library(RColorBrewer)
library(clusterProfiler)
library(org.Mm.eg.db)
library(AnnotationDbi)

exp = read_csv("normalized_data.csv")
exp = discard(exp,~all(is.na(.) |.  == ""))
exp = distinct(exp, `...1`, .keep_all = TRUE)
# filtering out the reprogramming transcription factors 
exp = filter(exp, !`...1`%in% c("Mef2c", "Gata4", "Tbx5"))
exp = column_to_rownames(exp, var="...1")

seurat.obj = CreateSeuratObject(counts = exp)

seurat.obj = NormalizeData(seurat.obj)
seurat.obj = FindVariableFeatures(seurat.obj, selection.method =  "vst", nfeatures = 10000)
all.genes = rownames(seurat.obj)
seurat.obj = ScaleData(seurat.obj, features = all.genes)
seurat.obj = RunPCA(seurat.obj, features =  VariableFeatures(seurat.obj))

ElbowPlot(seurat.obj)

seurat.obj = FindNeighbors(seurat.obj, dims = 1:15)

Computing nearest neighbor graph
Computing SNN

seurat.obj = FindClusters(seurat.obj, resolution = 1.75)

Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 454
Number of edges: 16805

Running Louvain algorithm...

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[----|----|----|----|----|----|----|----|----|----|
**************************************************|

Maximum modularity in 10 random starts: 0.4797
Number of communities: 8
Elapsed time: 0 seconds

identified_cells = read.csv("paper_cell_states.csv", row.names = 1, header=TRUE)

identified_cells = identified_cells[match(rownames(seurat.obj@meta.data), rownames(identified_cells)),]
seurat.obj@meta.data$cc_paper = identified_cells$CC
seurat.obj@meta.data$cat_paper = identified_cells$stage
seurat.obj@meta.data$cc_hclust = "CCI"
seurat.obj@meta.data[active_cells,]$cc_hclust = "CCA"

Idents(seurat.obj)  = "RNA_snn_res.1.75"

seurat.obj = RunUMAP(seurat.obj, dims = 1:15)

Warning: The default method for RunUMAP has changed from calling Python UMAP via reticulate to the R-native UWOT using the cosine metric
To use Python UMAP via reticulate, set umap.method to 'umap-learn' and metric to 'correlation'
This message will be shown once per session
17:59:25 UMAP embedding parameters a = 0.9922 b = 1.112
17:59:25 Read 454 rows and found 15 numeric columns
17:59:25 Using Annoy for neighbor search, n_neighbors = 30
17:59:25 Building Annoy index with metric = cosine, n_trees = 50
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**************************************************|
17:59:25 Writing NN index file to temp file /var/folders/fj/09brpj8s5mz1c5wxktl80s080000gn/T//RtmpE0XJwn/file8ed51e4226b8
17:59:25 Searching Annoy index using 1 thread, search_k = 3000
17:59:25 Annoy recall = 100%
17:59:26 Commencing smooth kNN distance calibration using 1 thread with target n_neighbors = 30
17:59:27 Initializing from normalized Laplacian + noise (using irlba)
17:59:27 Commencing optimization for 500 epochs, with 17028 positive edges
Using method 'umap'
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**************************************************|
17:59:28 Optimization finished

clusters = DimPlot(seurat.obj, group.by = "RNA_snn_res.1.75", label = TRUE, reduction = "umap")

condition = DimPlot(seurat.obj, group.by = "cc_paper", label = TRUE, reduction = "umap")

condition|clusters

seurat.obj = RunTSNE(seurat.obj, dims = 1:15)
DimPlot(seurat.obj, reduction = "tsne", group.by = 'RNA_snn_res.1.75')

markers = FindAllMarkers(seurat.obj, logfc.threshold =  0.25, min.pct = 0.1, only.pos = FALSE, test.use = 'MAST')

top_markers = markers %>%
    group_by(cluster) %>%
    slice_max(n = 2, order_by = avg_log2FC)

FeaturePlot(seurat.obj, features = c("Dhrs3"), min.cutoff = 'q10')

VlnPlot(seurat.obj, features = c("Rbp1", "Dhrs3"))

VlnPlot(seurat.obj, features = c("Fstl1", "Bgn"))

VlnPlot(seurat.obj, features = c("Serpinh1", "Dsp"))

VlnPlot(seurat.obj, features = c("Prc1", "Birc5"))

## cell cycle 
s.genes <- str_to_title(cc.genes$s.genes)
g2m.genes <- str_to_title(cc.genes$g2m.genes)

seurat.obj = CellCycleScoring(seurat.obj, s.features = s.genes, g2m.features = g2m.genes)

clusters = DimPlot(seurat.obj, group.by = "RNA_snn_res.1.75", label = TRUE, reduction = "umap")

real_condition = DimPlot(seurat.obj, group.by = "cc_paper", label = TRUE, reduction = "umap") 

phase = DimPlot(seurat.obj, group.by = "Phase", label = TRUE, reduction = "umap") 

cat = DimPlot(seurat.obj, group.by = "cc_hclust", label = TRUE, reduction = "umap") 

clusters|real_condition|phase|cat

NA
NA

subsetting the cells that fit with hclust and performing clustering on each group

subset(x = seurat.obj, subset = cc_hclust == "CCA")

An object of class Seurat 
14410 features across 80 samples within 1 assay 
Active assay: RNA (14410 features, 10000 variable features)
 3 dimensional reductions calculated: pca, umap, tsne

clustering CCI cels

cci_seurat = FindNeighbors(cci_seurat, dims = 1:15)
cci_seurat = FindClusters(cci_seurat, resolution = 1)

cl= DimPlot(cci_seurat, group.by = "RNA_snn_res.1",label = TRUE)

pap = DimPlot(cci_seurat, group.by = "cat_paper", label = TRUE)

cl|pap

identifying CCI cells

cci_markers = FindAllMarkers(cci_seurat, logfc.threshold =  0.25, min.pct = 0.1, only.pos = FALSE, test.use = 'MAST')

cci_top_markers = cci_markers %>%
    group_by(cluster) %>%
    slice_min(n = 5, order_by = p_val_adj)

VlnPlot(cci_seurat, features = c("Fstl1", "Bgn"))

VlnPlot(cci_seurat, features = c("Tnni3", "Smpx"))

VlnPlot(cci_seurat, features = c("Serpinh1", "Dsp"))

VlnPlot(cci_seurat, features = c("Clca2", "Rbp1"))

VlnPlot(cci_seurat, features = c("Cfh", "B4galt1"))

Fstl1 involved in endothelial cell differentiation and endothelial cell migration and Bgn is a gene that is used for connective tissue metabolism -> lower expression -> cluster 0 = iCM

Tnni3, smpx, dsp used for cardiomycoytes -> cluster 2 = pre-iCM, cluster 1 and 3 are still fibroblasts

cluseter 1 further away from cluster 0 = fibroblastr clusetr 3 = induced fibroblasts

Idents(cci_seurat)  = "RNA_snn_res.1"
cci_seurat = RenameIdents(cci_seurat, `0` = "iCM")
cci_seurat = RenameIdents(cci_seurat, `1` = "Fib")
cci_seurat = RenameIdents(cci_seurat, `2` = "piCM")
cci_seurat = RenameIdents(cci_seurat, `3` = "iFib")

clustering CCA cells

cca_seurat = FindClusters(cca_seurat, resolution = 1)

Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 80
Number of edges: 2244

Running Louvain algorithm...

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[----|----|----|----|----|----|----|----|----|----|
**************************************************|

Maximum modularity in 10 random starts: 0.2948
Number of communities: 3
Elapsed time: 0 seconds

cl= DimPlot(cca_seurat, group.by = "RNA_snn_res.1",label = TRUE)

pap = DimPlot(cca_seurat, group.by = "cat_paper", label = TRUE)

cl|pap

identifying CCA cells

cca_markers = FindAllMarkers(cca_seurat, logfc.threshold =  0.25, min.pct = 0.1, only.pos = FALSE, test.use = 'MAST')

Calculating cluster 0

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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...

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 Completed [=======================================================================================================] 100% with 0 failures
                                                                                                                                         

Done!
Calculating cluster 1

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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...

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 Completed [=======================================================================================================] 100% with 0 failures
                                                                                                                                         

Done!
Calculating cluster 2

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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...

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Done!

cca_top_markers = cca_markers %>%
    group_by(cluster) %>%
    slice_min(n = 5, order_by = p_val_adj)

VlnPlot(cca_seurat, features = c("Dab2", "Kcnab1"))

VlnPlot(cca_seurat, features = c("Cenpf", "Pttg1"))

VlnPlot(cca_seurat, features = c("Upk1b", "Timp3"))

NA
NA

dab2 involved in endothelial stuf -> 0 is probably induced cardiomyocyet Upk1b is involved in epithelial stuff -> 2 is porbably fibroblasts 1 - induced fibroblasts

Idents(cca_seurat)  = "RNA_snn_res.1"
cca_seurat = RenameIdents(cca_seurat, `0` = "iCM")
cca_seurat = RenameIdents(cca_seurat, `1` = "iFib")
cca_seurat = RenameIdents(cca_seurat, `2` = "Fib")

DimPlot(cca_seurat, label = TRUE)

SC Transform

seurat.sc = CreateSeuratObject(counts = exp)
seurat.sc = SCTransform(seurat.sc)

seurat.sc = RunPCA(seurat.sc, features =  VariableFeatures(seurat.sc))

identified_cells = identified_cells[match(rownames(seurat.sc@meta.data), rownames(identified_cells)),]
seurat.sc@meta.data$cc_paper = identified_cells$CC
seurat.sc@meta.data$cat_paper = identified_cells$stage

seurat.sc = RunUMAP(seurat.sc, dims = 1:15)
seurat.sc = FindNeighbors(seurat.sc, dims = 1:15)
seurat.sc = FindClusters(seurat.sc, resolution = 1.75)

seurat.sc = CellCycleScoring(seurat.sc, s.features = s.genes, g2m.features = g2m.genes)

clusters = DimPlot(seurat.sc, group.by = "SCT_snn_res.1.75", label = TRUE, reduction = "umap")
real_condition = DimPlot(seurat.sc, group.by = "cc_paper", label = TRUE, reduction = "umap") 
phase = DimPlot(seurat.sc, group.by = "Phase", label = TRUE, reduction = "umap") 
cat = DimPlot(seurat.sc, group.by = "cat_paper", label = TRUE, reduction = "umap") 

clusters|real_condition|phase|cat